The insulin polypeptide is the primary hormone responsible for controlling the transport, utilization and storage of glucose in the body. The β-cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin. Proteolysis of proinsulin results in removal of certain basic amino acids in the proinsulin chain along with the connecting peptide (C-peptide) to yield the biologically active polypeptide insulin.
The insulin molecule has been highly conserved in evolution and generally consists of two chains of amino acids linked by disulfide bonds. In the natural human, two-chain insulin molecule (mw 5,800 Daltons), the A-chain is composed of 21 amino acid residues and has glycine at the amino terminus and the B-chain has 30 amino acid residues and phenylalanine at the amino terminus.
Insulin can exist as a monomer or may aggregate into a dimer or a hexamer formed from three of the dimers. Biological activity, i.e., the ability to bind to receptors and stimulate the biological actions of insulin, resides in the monomer.
Diabetes is a biological disorder involving improper carbohydrate metabolism. Diabetes results from insufficient production of, or reduced sensitivity to, insulin. In persons with diabetes, the normal ability to use glucose is inhibited, leading to elevated blood sugar levels (hyperglycemia). As glucose accumulates in the blood, excess levels of sugar are excreted in the urine (glycosuria). Other symptoms of diabetes include increased urinary volume and frequency, thirst, itching, hunger, weight loss, and weakness.
There are two varieties of diabetes. Type I is insulin-dependent diabetes mellitus, or IDDM. IDDM was formerly referred to as “juvenile onset diabetes.” In IDDM, insulin is not secreted by the pancreas and must be provided from an external source. Type II or adult-onset diabetes can ordinarily be controlled by diet, although in some advanced cases, administration of insulin is required.
Untreated diabetes leads to ketosis, the accumulation of ketones, which are products of fat breakdown, in the blood. Ketosis is followed by the accumulation of acid in the blood (acidosis), nausea and vomiting. As the toxic products of disordered carbohydrate and fat metabolism continue to build up, the patient goes into a diabetic coma, which leads to death. Before the isolation of insulin in the 1920s, most patients died within a short time after onset.
The use of insulin as a treatment for diabetes dates to 1922, when Banting et al. (“Pancreatic Extracts in the Treatment of Diabetes Mellitus,” Can. Med. Assoc. J., 12:141–146 (1922)) showed that the active extract from the pancreas had therapeutic effects in diabetic dogs. In that same year, treatment of a diabetic patient with pancreatic extracts resulted in a dramatic, life-saving clinical improvement.
Until recently, bovine and porcine insulin were used almost exclusively to treat diabetes in humans. Today, however, numerous variations in insulin between species are known. Each variation differs from natural human insulin in having amino acid substitution(s) at one or more positions in the A- and/or B-chain. Despite these differences, most mammalian insulin has comparable biological activity. The advent of recombinant technology has enabled commercial scale manufacture of human insulin (e.g., Humulin™ insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.) or genetically engineered insulin having biological activity comparable to natural human insulin.
Treatment of diabetes typically requires regular injections of insulin. Due to the inconvenience of insulin injections, massive efforts to improve insulin administration and bioassimilation have been undertaken.
Attempts have been made to deliver insulin by oral administration. The problems associated with oral administration of insulin to achieve euglycemia in diabetic patients are well documented in pharmaceutical and medical literature. Digestive enzymes in the gastrointestinal tract rapidly degrade insulin, resulting in biologically inactive breakdown products. In the stomach, for example, orally administered insulin undergoes enzymatic proteolysis and acidic degradation. Comparable proteolytic breakdown of insulin occurs in the intestine. In the lumen, insulin is attacked by a variety of enzymes including gastric and pancreatic enzymes, exo- and endopeptidases, and brush border peptidases. Even if insulin survives this enzymatic attack, the biological barriers that must be traversed before insulin can reach its receptors in vivo can limit its bioavailability after oral administration of insulin. For example, insulin can possess low membrane permeability, limiting its ability to pass from the intestinal lumen into the bloodstream.
Some efforts to provide an oral form of insulin have focused on providing insulin-oligomer conjugates. Human insulin and many closely related insulins that are used therapeutically contain three amino acid residues bearing free primary amino groups. All three primary amino groups, namely the N-termini (alpha amino groups) of the A and B chains (GlyA1 and PheB1) and the epsilon-amino group of LysB29, can be modified by conjugation with oligomers. Depending on the reaction conditions, N-acylation of an unprotected insulin leads to a complex mixture of mono-, di-, and tri-conjugates (e.g., insulin mono-conjugated at GlyA1, insulin mono-conjugated at PheB1, insulin mono-conjugated at LysB29, insulin di-conjugated at GlyA1 and PheB1, insulin di-conjugated at GlyA1 and LysB29, insulin di-conjugated at PheB1 and LysB29, and insulin tri-conjugated at GlyA1, PheB1, and LysB29). When a particular conjugate, for example insulin mono-conjugated at LysB29, is desired, it can be burdensome and/or expensive to separate (or purify) such a complex mixture of conjugates to obtain the desired conjugate.
As a result, various efforts have been undertaken to selectively synthesize the desired insulin conjugate. For example, Muranishi and Kiso, in Japanese Patent Application 1-254,699, propose a five-step synthesis for preparing fatty acid insulin derivatives. The A1- and B1-amino groups of insulin are protected (or blocked) with p-methoxybenzoxy carbonyl azide (pMZ). After acylation with a fatty acid ester, the protection (blocking) groups are removed to provide insulin mono-acylated at Lys(B29) with a fatty acid. As another example, U.S. Pat. No. 5,750,497 to Havelund et al. proposes treating human insulin with a Boc-reagent (e.g. di-tert-butyl dicarbonate) to form (A1,B1)-diBoc human insulin, i.e., human insulin in which the N-terminal end of both the A- and B-chains are protected by a Boc-group. After an optional purification, e.g., by HPLC, a lipophilic acyl group is introduced in the ε-amino group of LysB29 by allowing the product to react with a N-hydroxysuccinimide ester of the formula X-OSu wherein X is the lipophilic acyl group to be introduced. In the final step, trifluoroacetic acid is used to remove the Boc-groups and the product, NεB29-X human insulin, is isolated.
Various other efforts have been undertaken to preferentially synthesize the desired insulin conjugate to provide a mixture of conjugates in which the desired insulin conjugate is the preferred product. For example, U.S. Pat. No. 5,646,242 to Baker et al. proposes a reaction that is performed without the use of amino-protecting groups. Baker proposes the reaction of an activated fatty ester with the ε-amino group of insulin under basic conditions in a polar solvent. The acylation of the ε-amino group is dependent on the basicity of the reaction. At a pH greater than 9.0, the reaction preferentially acylates the ε-amino group of B29-lysine over the α-amino groups. Examples 1 through 4 report reaction yields of the mono-conjugated insulin as a percentage of the initial amount of insulin between 67.1% and 75.5%. In Example 5, Baker also proposes acylation of human proinsulin with N-succinimidyl palmitate. The exact ratios of ε-amino acylated species to α-amino acylated species were not calculated. The sum of all ε-amino acylated species within the chromatogram accounted for 87–90% of the total area, while the sum of all related substances (which would presumably include any α-amino acylated species) accounted for <7% of the total area, for any given point in time.
The present invention overcomes previous limitations in the art by providing methods of site-specifically synthesizing particular insulin-oligomer conjugates that are less burdensome and/or more cost effective than conventional methods.